Treatment of cancer with dual targeting of CD47 and EGFR

ABSTRACT

Methods are provided for targeting cells for depletion, including without limitation cancer cells, in a regimen comprising contacting the targeted cells with a combination of agents, including (i) an agent that blockades CD47 activity; and (ii) an antibody that specifically binds to EGFR. In some embodiments the cancer cells have a mutated form of one or more of KRAS, NRAS or BRAF. The level of depletion of the targeted cell is enhanced relative to a regimen in which a single agent is used; and the effect may be synergistic relative to a regimen in which a single agent is used.

CROSS REFERENCE

This application claims benefit of U.S. Provisional Patent ApplicationNo. 62/380,177, filed Aug. 26, 2016, and also claims benefit of U.S.Provisional Patent Application No. 62/266,470, filed Dec. 11, 2015,which applications are incorporated herein by reference in theirentirety.

FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under contract CA139490awarded by the National Institutes of Health. The Government has certainrights in the invention.

Antibody-based therapy for cancer has become established as a successfuland important strategy for treating patients with hematologicalmalignancies and solid tumors. The definition of cell surface antigensthat are expressed by human cancers has revealed a broad array oftargets that are overexpressed, mutated or selectively expressedcompared with normal tissues. A key challenge has been to identifyantigens that are suitable for antibody-based therapeutics. Suchtherapeutics can function through mediating alterations in antigen orreceptor function (such as agonist or antagonist functions), modulatingthe immune system or delivering a specific drug that is conjugated to anantibody that targets a specific antigen.

Depending on the antibody agent, killing of cancer cells can be theresult of receptor blockade or agonist activity, induction of apoptosis,delivery of a drug or cytotoxic agent; immune-mediated cell killingmechanisms including, complement-dependent cytotoxicity (CDC),antibody-dependent cellular cytotoxicity (ADCC) and regulation of T cellfunction; and specific effects of an antibody on tumor vasculature andstroma. Antibodies that inhibit growth factors or growth factorreceptors in cancer patients include CEA, epidermal growth factorreceptor (EGFR; also known as ERBB1), ERBB2 (also known as HER2), ERBB3,MET (also known as HGFR), insulin-like growth factor 1 receptor (IGF1R),ephrin receptor A3 (EPHA3), tumor necrosis factor (TNF)-relatedapoptosis-inducing ligand receptor 1 (TRAILR1; also known as TNFRSF10A),TRAILR2 (also known as TNFRSF10B) and receptor activator of nuclearfactor-KB ligand (RANKL; also known as TNFSF11).

Several anti-EGFR antibodies are currently in clinical use. Vectibix®(panitumumab) is an epidermal growth factor receptor antagonist approvedas a single agent for the treatment of metastatic colorectal carcinomawith disease progression on or following chemotherapy regimens. Erbitux®(cetuximab) is an epidermal growth factor receptor (EGFR) antagonistapproved for treatment of locally or regionally advanced squamous cellcarcinoma of the head and neck; and as a single agent or combined withirinotecan for treatment of EGFR-expressing metastatic colorectalcancer. Nimotuzumab is a humanized IgG antibody against EGFR approvedfor use in some countries in Asia, South American and Africa for thetreatment of head and neck cancer, glioma and nasopharyngeal cancer.

Antibodies that disrupt growth factor signaling, however, can beineffective in the treatment of tumors where the signaling pathway ismutated. For example, many colorectal cancers carry mutations in theEGFR pathway that result in constitutive activation. Recent evidence hasshown that patients with mutations in KRAS or NRAS do not respond toanti-EGFR antibodies, resulting in the approved use of these agentsbeing restricted to patients with cancer in which KRAS is not mutated.The use of trastuzumab has also been restricted to patients with highlevels of ERBB2 expression, as studies have shown that this is the groupthat derives maximum benefit from trastuzumab treatment.

Aside from targeting antigens that are involved in cancer cellproliferation and survival, antibodies can also function to eitheractivate or antagonize immunological pathways that are important incancer immune surveillance. It is now clear that an antigen-specificimmune response is the result of a complex dynamic interplay betweenantigen-presenting cells, phagocytes, T lymphocytes and target cells.

For example, the cell surface protein CD47 on healthy cells and itsengagement of a phagocyte receptor, SIRPα, constitutes a key “don'teat-me” signal that can turn off engulfment mediated by multiplemodalities, including apoptotic cell clearance and FcR mediatedphagocytosis. Blocking the CD47 mediated engagement of SIRPα on aphagocyte, or the loss of CD47 expression in knockout mice, can causeremoval of live cells and non-aged erythrocytes. Alternatively, blockingSIRPα recognition also allows engulfment of targets that are notnormally phagocytosed.

Related publications include U.S. Pat. No. 8,728,476; Dalerba, P., S. J.Dylla, Et Al. (2007). “Phenotypic Characterization Of Human ColorectalCancer Stem Cells.” Proc Natl Acad Sci USA 104(24): 10158-10163;Weiskopf, K., A. M. Ring, Et Al. (2013). “Engineered Sirp alpha VariantsAs Immunotherapeutic Adjuvants To Anticancer Antibodies.” Science341(6141): 88-91; Willingham, S. B., J. P. Volkmer, Et Al. (2012). “TheCd47-Signal Regulatory Protein Alpha (Sirpa) Interaction Is ATherapeutic Target For Human Solid Tumors.” Proc Natl Acad Sci USA109(17): 6662-6667. “Epigentic and genetic features of 24 colon cancercell lines”, Ahmed et al., Oncogenesis, 2013; Chao, M. P., A. A.Alizadeh, Et Al. (2010). “Anti-Cd47 Antibody Synergizes Wth Rituximab ToPromote Phagocytosis And Eradicate Non-Hodgkin Lymphoma.” Cell 142(5):699-713.

SUMMARY OF THE INVENTION

Methods are provided for improved treatment of epidermal growth factorreceptor (EGFR) expressing cancers in an individual, which cancersinclude without limitation any EGFR-expressing cancer, e.g. carcinomassuch as adenocarcinomas, squamous cell carcinomas, basal cellcarcinomas, renal cell carcinomas; and gliomas. Cancers of interest mayinclude colorectal carcinomas, non-small cell lung carcinoma (NSCLC),ovarian cancer, pancreatic cancer, breast cancer, squamous cellcarcinomas, and gliomas. In the methods of the invention, cancer cellsare contacted with a combination of (a) an agent that blocks signalingbetween CD47 and SIRPα; and (b) an antibody that specifically binds toEGFR. The methods of the invention can provide for increased overallsurvival of the individual being treated, in contrast to treatment withknown EGFR antagonist antibodies in the absence of CD47 blockade.

In some embodiments the EGFR-expressing cancer cells comprise a mutationthat results in constitutive activation of the EGFR signaling pathway.In some such embodiments the mutation is a KRAS, NRAS or BRAF mutationin one or both alleles. In some embodiments the mutation is in codon 12or 13 of KRAS. Such individuals are excluded from therapy with anti-EGFRantagonist antibodies, but are shown herein to benefit from treatmentwith the combination therapy of the present invention. In otherembodiments individuals having wild-type KRAS, NRAS, BRAF are treated bythe methods of the invention.

Individuals may be selected for therapy by determining the genotype ofthe cancer cells with respect to one or more of KRAS, NRAS and BRAF.Individuals currently excluded from therapy with anti-EGFR antibodiesdue to the presence of a mutation in KRAS or NRAS can be selected fortreatment with the methods of the present invention. Individuals mayalso be tested for the expression of detectable EGFR on the cancercells, where cancers showing positive expression of EGFR are selectedfor treatment.

The agents in the combination are administered concomitantly, i.e. eachagent is administered within about 45 days, 30 days, 15 days, 7 days, 3days, 2 days, 1 day or substantially simultaneously with respect to theother agent(s) in the combination. The agents can be considered to becombined if administration scheduling is such that the serum level ofboth agents is at a therapeutic level. A benefit of the presentinvention can be the use of lowered doses of the anti-EGFR antibodyrelative to the dose required as a monotherapy. Administration may berepeated as necessary for depletion of the cancer cell population.

In some embodiments a primer agent is administered prior toadministering a therapeutically effective dose of an anti-CD47 agent tothe individual. Suitable primer agents include anerythropoiesis-stimulating agent (ESA), and/or a priming dose of ananti-CD47 agent. Following administration of the priming agent, andallowing a period of time effective for an increase in reticulocyteproduction, a therapeutic dose of an anti-CD47 agent is administered.The therapeutic dose can be administered in number of different ways. Insome embodiments, two or more therapeutically effective doses areadministered after a primer agent is administered. In some embodiments atherapeutically effective dose of an anti-CD47 agent is administered astwo or more doses of escalating concentration, in others the doses areequivalent.

In some embodiments, administration of a combination of agents of theinvention is combined with an effective dose of an agent that increasespatient hematocrit, for example erythropoietin stimulating agents (ESA).Such agents are known and used in the art, including, for example,Aranesp® (darbepoetin alfa), Epogen®NF/Procrit®NF (epoetin alfa),Omontys® (peginesatide), Procrit®, etc.

An anti-CD47 agent for use in the methods of the invention interfereswith binding between CD47 present on the cancer cell and SIRPα presenton a phagocytic cell. Such methods, in the presence of the anti-EGFRantibody, can increase phagocytosis of the cancer cell. Suitableanti-CD47 agents include soluble SIRPα polypeptides; soluble CD47;anti-CD47 antibodies, anti-SIRPα antibodies, and the like, where theterm antibodies encompasses antibody fragments and variants thereof, asknown in the art. In some embodiments the anti-CD47 agent is ananti-CD47 antibody. In some embodiments the anti-CD47 antibody is anon-hemolytic antibody. In some embodiments the antibody comprises ahuman IgG4 Fc region.

Anti-EGFR antibodies include, without limitation, antibodies in currentclinical use and in clinical trials, which include antibodies that actthrough antagonizing EGFR signaling. While such EGFR antagonistantibodies can be used for the methods of the invention, the methods ofthe invention do not require that the anti-EGFR antibody block EGFRsignaling, and thus antibodies that are not pathway antagonists can beused in the present combination therapy methods. Examples of anti-EGFRantibodies currently approved for clinical use include the chimericmonoclonal antibody cetuximab; which contains the murine variable regionof mAb225 and a human IgG1 constant region; the fully human IgG2antibody panitumumab; and the humanized IgG1 antibody nimotuzumab. Otherantibodies in clinical development include zalutumumab and matuzumab.The anti-EGFR antibody may be administered in accordance withconventional dosing suitable as a monotherapy or combined withchemotherapy and/or radiation; or the dosage may be adjusted to optimizethe effectiveness when combined with CD47 blockade.

The contacting of a cancer cells may be performed in vivo, e.g. fortherapeutic purposes, and in vitro, e.g. for screening assays and thelike. Tumor cells, e.g. carcinomas, gliomas, melanomas, etc. aretargeted for depletion by contacting the immune cells, includingphagocytic cells, in proximity of the tumor cells with a combination ofa CD47 blocking agent that is effective to block the interaction betweenCD47 and SIRPα, and an anti-EGFR antibody. The combination therapy canbe synergistic in enhancing phagocytosis and elimination of tumor cellsas compared to the use of single agents. The combination may also inducean anti-tumor T cell response. Further, the combination can provide forincreased overall survival of the individual that is treated.

BRIEF DESCRIPTION OF THE FIGURES

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. The patent orapplication file contains at least one drawing executed in color. Copiesof this patent or patent application publication with color drawing(s)will be provided by the Office upon request and payment of the necessaryfee. It is emphasized that, according to common practice, the variousfeatures of the drawings are not to-scale. On the contrary, thedimensions of the various features are arbitrarily expanded or reducedfor clarity. Included in the drawings are the following figures.

FIG. 1. Expression of EGFR on cancer stem cells. Patient colon cancercells were profiled for markers indicative of cancer stem cells (CD44+CD166+) as well as expression of CD47 and EGFR. It is shown that most ifnot all CSCs co-express EGFR.

FIG. 2. Blockade of the CD47-SIRPα pathway (anti-CD47 Ab, Hu5F9-G4)enhances antibody dependent cellular phagocytosis (ADCP) ofEGFR-expressing cancer cells by anti-EGFR antibodies (cetuximab)irrespective of downstream mutations in the EGFR signaling pathway.Phagocytosis of a panel of colon cancer cells by donor-derived humanmacrophages in the presence of control IgG4 (black), anti-CD47 AbHu5F9-G4 (red), anti-EGFR Ab cetuximab (green), or combination ofHu5F9-G4 with cetuximab (blue). Mutational status of KRAS and BRAF aspreviously reported (Epigentic and genetic features of 24 colon cancercell lines, Ahmed et al., Oncogenesis, 2013). Cells are organized byincreasing expression of EGFR; SW620 is negative for EGFR expression asdetermined by cetuximab binding by flow cytometry. Data represent meanand standard deviation using macrophages from 2 independent blooddonors.

FIG. 3. Combination of anti-EGFR antibodies (cetuximab, panitumumab)with blockade of the CD47-SIRPα signaling pathway by anti-CD47 antibody(Hu5F9-G4) enhances therapeutic efficacy in vivo. (A). Tumor Growth. UM8colon adenocarcinoma cells (EGFR⁺) transduced with GFP-luciferaseencoding lentivirus were subcutaneously injected into the backs of NSGmice. Twenty-eight days after tumor cell engraftment (confirmed bybioluminescence imaging), PBS (black), Hu5F9-G4 (red) (250 μg),cetuximab (green 120 μg), panitumumab (blue) (120 μg), Hu5F9-G4 incombination with cetuximab (orange), or Hu5F9-G4 in combination withpanitumumab (purple) were administered via IP injection every other day(PBS, Hu5F9-G4) or weekly (panitumumab, cetuximab) for thirteen weeks.Tumor growth was measured by bioluminescence imaging. Administration ofHu5F9-G4 alone did not inhibit tumor growth, and treatment withcetuximab monotherapy stabilized tumor growth but failed to produce atumor regression. Treatment with panitumumab monotherapy induced a minortumor regression while a major tumor regression was observed in all micetreated with Hu5F9-G4 in combination with cetuximab or panitumumab. (B).Survival Curve.

FIG. 4. Combination of anti-EGFR antibodies (cetuximab) with blockade ofthe CD47-SIRPα signaling pathway by anti-CD47 antibody (Hu5F9-G4)enhances antibody dependent cellular phagocytosis (ADCP) ofEGFR-expressing cancer cells irrespective of downstream mutations in theEGFR signaling pathway and prevents or eliminates metastasis. (A). DLD1colon adenocarcinoma cells (EGFR⁺⋅KRAS mutant) transduced withGFP-luciferase encoding lentivirus were subcutaneously injected into thebacks of NSG mice. Five days after tumor cell engraftment (confirmed bybioluminescence imaging), PBS (black), Hu5F9-G4 (red) (250 μg),cetuximab (green) (120 μg), or Hu5F9-G4 in combination with cetuximab(blue) were administered via IP injection every other day (PBS,Hu5F9-G4) or weekly (cetuximab) for seven weeks. Tumor growth wasmeasured by bioluminescence imaging. Administration of Hu5F9-G4 aloneslowed but did not inhibit tumor growth. Treatment with cetuximabmonotherapy had no effect as expected for tumors with downstreammutations in the EGFR signaling pathway. Hu5F9-G4 in combination withcetuximab did not inhibit tumor growth but had the strongest effect toslow down tumor growth. (B-C). Remarkably, mice treated with cetuximabhad less lymph node (4/5 mice) and lung metastasis (1/5 mice) comparedto PBS control cohort (LN 4/5 and lung 4/5) but the therapeutic effectwas less potent than with Hu5F9-G4 (LN 2/4 and lung 0/4). The strongesttherapeutic effect had the combination Hu5F9-G4 with cetuximab. None ofthe mice in this cohort had any metastasis at the end of treatment (LN0/5 and lung 0/5).

FIG. 5. Schematic of clinical trial protocol, showing dosing and timingof combination.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Methods are provided for the targeted depletion of EGFR-expressingcancer cells in a subject, where the cancer cells are selectivelyablated by phagocytosis of the living cells, following contacting with acombination of agents that (a) block CD47 signaling; and (b) targetEGFR.

To facilitate an understanding of the invention, a number of terms aredefined below.

Before the present active agents and methods are described, it is to beunderstood that this invention is not limited to the particularmethodology, products, apparatus and factors described, as such methods,apparatus and formulations may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention which will be limited only by appendedclaims.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “and,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “adrug candidate” refers to one or mixtures of such candidates, andreference to “the method” includes reference to equivalent steps andmethods known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications mentionedherein are incorporated herein by reference for the purpose ofdescribing and disclosing devices, formulations and methodologies whichare described in the publication and which might be used in connectionwith the presently described invention.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges is also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either both ofthose included limits are also included in the invention.

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of skill in the art that the presentinvention may be practiced without one or more of these specificdetails. In other instances, well-known features and procedures wellknown to those skilled in the art have not been described in order toavoid obscuring the invention.

Generally, conventional methods of protein synthesis, recombinant cellculture and protein isolation, and recombinant DNA techniques within theskill of the art are employed in the present invention. Such techniquesare explained fully in the literature, see, e.g., Maniatis, Fritsch &Sambrook, Molecular Cloning: A Laboratory Manual (1982); Sambrook,Russell and Sambrook, Molecular Cloning: A Laboratory Manual (2001);Harlow, Lane and Harlow, Using Antibodies: A Laboratory Manual: PortableProtocol No. I, Cold Spring Harbor Laboratory (1998); and Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory;(1988).

Definitions

Anti-CD47 agent. CD47 is a broadly expressed transmembrane glycoproteinwith a single Ig-like domain and five membrane spanning regions, whichfunctions as a cellular ligand for SIRPα with binding mediated throughthe NH2-terminal V-like domain of SIRPα. SIRPα is expressed primarily onmyeloid cells, including macrophages, granulocytes, myeloid dendriticcells (DCs), mast cells, and their precursors, including hematopoieticstem cells. Structural determinants on SIRPα that mediate CD47 bindingare discussed by Lee et al. (2007) J. Immunol. 179:7741-7750; Hatherleyet al. (2008) Mol Cell. 31(2):266-77; Hatherley et al. (2007) J.B.C.282:14567-75; and the role of SIRPα cis dimerization in CD47 binding isdiscussed by Lee et al. (2010) J.B.C. 285:37953-63. In keeping with therole of CD47 to inhibit phagocytosis of normal cells, there is evidencethat it is transiently upregulated on hematopoietic stem cells (HSCs)and progenitors just prior to and during their migratory phase, and thatthe level of CD47 on these cells determines the probability that theyare engulfed in vivo.

As used herein, the term “anti-CD47 agent” or “agent that provides forCD47 blockade” refers to any agent that reduces the binding of CD47(e.g., on a target cell) to SIRPα (e.g., on a phagocytic cell).Non-limiting examples of suitable anti-CD47 reagents include SIRPαreagents, including without limitation high affinity SIRPα polypeptides,anti-SIRPα antibodies, soluble CD47 polypeptides, and anti-CD47antibodies or antibody fragments. In some embodiments, a suitableanti-CD47 agent (e.g. an anti-CD47 antibody, a SIRPα reagent, etc.)specifically binds CD47 to reduce the binding of CD47 to SIRPα.

In some embodiments, a suitable anti-CD47 agent (e.g., an anti-SIRPαantibody, a soluble CD47 polypeptide, etc.) specifically binds SIRPα toreduce the binding of CD47 to SIRPα. A suitable anti-CD47 agent thatbinds SIRPα does not activate SIRPα (e.g., in the SIRPα-expressingphagocytic cell). The efficacy of a suitable anti-CD47 agent can beassessed by assaying the agent. In an exemplary assay, target cells areincubated in the presence or absence of the candidate agent and in thepresence of an effector cell, e.g. a macrophage or other phagocyticcell. An agent for use in the methods of the invention will up-regulatephagocytosis by at least 5% (e.g., at least 10%, at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 100%, at least 120%, at least 140%, at least160%, at least 180%, at least 200%, at least 500%, at least 1000%)compared to phagocytosis in the absence of the agent. Similarly, an invitro assay for levels of tyrosine phosphorylation of SIRPα will show adecrease in phosphorylation by at least 5% (e.g., at least 10%, at least15%, at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 90%, or 100%) compared tophosphorylation observed in absence of the candidate agent.

In some embodiments, the anti-CD47 agent does not activate CD47 uponbinding. When CD47 is activated, a process akin to apoptosis (i.e.,programmed cell death) may occur (Manna and Frazier, Cancer Research,64, 1026-1036, Feb. 1, 2004). Thus, in some embodiments, the anti-CD47agent does not directly induce cell death of a CD47-expressing cell.

In some embodiments a primer agent is administered prior toadministering a therapeutically effective dose of an anti-CD47 agent tothe individual. Suitable primer agents include anerythropoiesis-stimulating agent (ESA), and/or a priming dose of ananti-CD47 agent. Following administration of the priming agent, andallowing a period of time effective for an increase in reticulocyteproduction, a therapeutic dose of an anti-CD47 agent is administered.Administration may be made in accordance with the methods described inco-pending patent application U.S. Ser. No. 14/769,069, hereinspecifically incorporated by reference.

SIRPα reagent. A SIRPα reagent comprises the portion of SIRPα that issufficient to bind CD47 at a recognizable affinity, which normally liesbetween the signal sequence and the transmembrane domain, or a fragmentthereof that retains the binding activity. A suitable SIRPα reagentreduces (e.g., blocks, prevents, etc.) the interaction between thenative proteins SIRPα and CD47. The SIRPα reagent will usually compriseat least the dl domain of SIRPα.

In some embodiments, a subject anti-CD47 agent is a “high affinity SIRPαreagent”, which includes SIRPα-derived polypeptides and analogs thereof(e.g., CV1-hIgG4, and CV1 monomer). High affinity SIRPα reagents aredescribed in international application PCT/US13/21937, which is herebyspecifically incorporated by reference. High affinity SIRPα reagents arevariants of the native SIRPα protein. The amino acid changes thatprovide for increased affinity are localized in the dl domain, and thushigh affinity SIRPα reagents comprise a dl domain of human SIRPα, withat least one amino acid change relative to the wild-type sequence withinthe dl domain. Such a high affinity SIRPα reagent optionally comprisesadditional amino acid sequences, for example antibody Fc sequences;portions of the wild-type human SIRPα protein other than the dl domain,including without limitation residues 150 to 374 of the native proteinor fragments thereof, usually fragments contiguous with the dl domain;and the like. High affinity SIRPα reagents may be monomeric ormultimeric, i.e. dimer, trimer, tetramer, etc. In some embodiments, ahigh affinity SIRPα reagent is soluble, where the polypeptide lacks theSIRPα transmembrane domain and comprises at least one amino acid changerelative to the wild-type SIRPα sequence, and wherein the amino acidchange increases the affinity of the SIRPα polypeptide binding to CD47,for example by decreasing the off-rate by at least 10-fold, at least20-fold, at least 50-fold, at least 100-fold, at least 500-fold, ormore.

Optionally the SIRPα reagent is a fusion protein, e.g., fused in framewith a second polypeptide. In some embodiments, the second polypeptideis capable of increasing the size of the fusion protein, e.g., so thatthe fusion protein will not be cleared from the circulation rapidly. Insome embodiments, the second polypeptide is part or whole of animmunoglobulin Fc region. The Fc region aids in phagocytosis byproviding an “eat me” signal, which enhances the block of the “don't eatme” signal provided by the high affinity SIRPα reagent. In otherembodiments, the second polypeptide is any suitable polypeptide that issubstantially similar to Fc, e.g., providing increased size,multimerization domains, and/or additional binding or interaction withIg molecules.

Anti-CD47 antibodies. In some embodiments, a subject anti-CD47 agent isan antibody that specifically binds CD47 (i.e., an anti-CD47 antibody)and reduces the interaction between CD47 on one cell (e.g., an infectedcell) and SIRPα on another cell (e.g., a phagocytic cell). In someembodiments, a suitable anti-CD47 antibody does not activate CD47 uponbinding. Some anti-CD47 antibodies do not reduce the binding of CD47 toSIRPα (and are therefore not considered to be an “anti-CD47 agent”herein) and such an antibody can be referred to as a “non-blockinganti-CD47 antibody.” A suitable anti-CD47 antibody that is an “anti-CD47agent” can be referred to as a “CD47-blocking antibody”. Non-limitingexamples of suitable antibodies include clones B6H12, 5F9, 8B6, and C3(for example as described in International Patent Publication WO2011/143624, herein specifically incorporated by reference). Suitableanti-CD47 antibodies include fully human, humanized or chimeric versionsof such antibodies. Humanized antibodies (e.g., hu5F9-G4) are especiallyuseful for in vivo applications in humans due to their low antigenicity.Similarly caninized, felinized, etc. antibodies are especially usefulfor applications in dogs, cats, and other species respectively.Antibodies of interest include humanized antibodies, or caninized,felinized, equinized, bovinized, porcinized, etc., antibodies, andvariants thereof.

In some embodiments an anti-CD47 antibody comprises a human IgG Fcregion, e.g. an IgG1, IgG2a, IgG2b, IgG3, IgG4 constant region. In apreferred embodiment the IgG Fc region is an IgG4 constant region. TheIgG4 hinge may be stabilized by the amino acid substitution S241P (seeAngal et al. (1993) Mol. Immunol. 30(1):105-108, herein specificallyincorporated by reference).

Anti-SIRPα antibodies. In some embodiments, a subject anti-CD47 agent isan antibody that specifically binds SIRPα (i.e., an anti-SIRPα antibody)and reduces the interaction between CD47 on one cell (e.g., an infectedcell) and SIRPα on another cell (e.g., a phagocytic cell). Suitableanti-SIRPα antibodies can bind SIRPα without activating or stimulatingsignaling through SIRPα because activation of SIRPα would inhibitphagocytosis. Instead, suitable anti-SIRPα antibodies facilitate thepreferential phagocytosis of inflicted cells over normal cells. Thosecells that express higher levels of CD47 (e.g., infected cells) relativeto other cells (non-infected cells) will be preferentially phagocytosed.Thus, a suitable anti-SIRPα antibody specifically binds SIRPα (withoutactivating/stimulating enough of a signaling response to inhibitphagocytosis) and blocks an interaction between SIRPα and CD47. Suitableanti-SIRPα antibodies include fully human, humanized or chimericversions of such antibodies. Humanized antibodies are especially usefulfor in vivo applications in humans due to their low antigenicity.Similarly caninized, felinized, etc. antibodies are especially usefulfor applications in dogs, cats, and other species respectively.Antibodies of interest include humanized antibodies, or caninized,felinized, equinized, bovinized, porcinized, etc., antibodies, andvariants thereof.

Soluble CD47 polypeptides. In some embodiments, a subject anti-CD47agent is a soluble CD47 polypeptide that specifically binds SIRPα andreduces the interaction between CD47 on one cell (e.g., an infectedcell) and SIRPα on another cell (e.g., a phagocytic cell). A suitablesoluble CD47 polypeptide can bind SIRPα without activating orstimulating signaling through SIRPα because activation of SIRPα wouldinhibit phagocytosis. Instead, suitable soluble CD47 polypeptidesfacilitate the preferential phagocytosis of infected cells overnon-infected cells. Those cells that express higher levels of CD47(e.g., infected cells) relative to normal, non-target cells (normalcells) will be preferentially phagocytosed. Thus, a suitable solubleCD47 polypeptide specifically binds SIRPα without activating/stimulatingenough of a signaling response to inhibit phagocytosis.

In some cases, a suitable soluble CD47 polypeptide can be a fusionprotein (for example as structurally described in US Patent PublicationUS20100239579, herein specifically incorporated by reference). However,only fusion proteins that do not activate/stimulate SIRPα are suitablefor the methods provided herein. Suitable soluble CD47 polypeptides alsoinclude any peptide or peptide fragment comprising variant or naturallyexisting CD47 sequences (e.g., extracellular domain sequences orextracellular domain variants) that can specifically bind SIRPα andinhibit the interaction between CD47 and SIRPα without stimulatingenough SIRPα activity to inhibit phagocytosis.

In certain embodiments, soluble CD47 polypeptide comprises theextracellular domain of CD47, including the signal peptide, such thatthe extracellular portion of CD47 is typically 142 amino acids inlength. The soluble CD47 polypeptides described herein also include CD47extracellular domain variants that comprise an amino acid sequence atleast 65%-75%, 75%-80%, 80-85%, 85%-90%, or 95%-99% (or any percentidentity not specifically enumerated between 65% to 100%), whichvariants retain the capability to bind to SIRPα without stimulatingSIRPα signaling.

In certain embodiments, the signal peptide amino acid sequence may besubstituted with a signal peptide amino acid sequence that is derivedfrom another polypeptide (e.g., for example, an immunoglobulin orCTLA4). For example, unlike full-length CD47, which is a cell surfacepolypeptide that traverses the outer cell membrane, the soluble CD47polypeptides are secreted; accordingly, a polynucleotide encoding asoluble CD47 polypeptide may include a nucleotide sequence encoding asignal peptide that is associated with a polypeptide that is normallysecreted from a cell.

In other embodiments, the soluble CD47 polypeptide comprises anextracellular domain of CD47 that lacks the signal peptide. As describedherein, signal peptides are not exposed on the cell surface of asecreted or transmembrane protein because either the signal peptide iscleaved during translocation of the protein or the signal peptideremains anchored in the outer cell membrane (such a peptide is alsocalled a signal anchor). The signal peptide sequence of CD47 is believedto be cleaved from the precursor CD47 polypeptide in vivo.

In other embodiments, a soluble CD47 polypeptide comprises a CD47extracellular domain variant. Such a soluble CD47 polypeptide retainsthe capability to bind to SIRPα without stimulating SIRPα signaling. TheCD47 extracellular domain variant may have an amino acid sequence thatis at least 65%-75%, 75%-80%, 80-85%, 85%-90%, or 95%-99% identical(which includes any percent identity between any one of the describedranges) to the native CD47 sequence.

EGFR. Epidermal growth factor receptor (EGFR) exists on the cell surfaceand is activated by binding of its specific ligands, including epidermalgrowth factor and transforming growth factor α (TGFα). Upon activationby its growth factor ligands, EGFR undergoes a transition from aninactive monomeric form to an active dimer. EGFR dimerization stimulatesits intrinsic intracellular protein-tyrosine kinase activity. As aresult, autophosphorylation of several tyrosine (Y) residues in theC-terminal domain of EGFR occurs. This autophosphorylation elicitsdownstream activation and signaling by several other proteins thatassociate with the phosphorylated tyrosines through their ownphosphotyrosine-binding SH2 domains. These downstream signaling proteinsinitiate several signal transduction cascades, principally the MAPK, Aktand JNK pathways, leading to DNA synthesis and cell proliferation.

Downstream effects of EGFR activation include modulation of three majorpathways. Induction of the RAS-RAF-MAPK pathway occurs whenphosphorylated EGFR recruits the guanine-nucleotide exchange factor viathe GRB2 and SHC adapter proteins, which activates RAS. This stepsubsequently stimulates RAF and the MAP kinase pathway, ultimatelyaffecting cell proliferation, tumor invasion, and metastasis. Theinvolvement of RAS in the EGFR signaling pathway is of importance fortreatment with anti-EGFR antagonist antibodies. EGFR inhibitors areeffective in only a small subset of patients, despite high levels ofEGFR expression. Some cancers appear to acquire resistance to EGFRinhibitors, and multiple mechanisms seem to underlie the lack ofsensitivity to the targeted therapies, including mutations in the EGFRgene itself, as well as in down-stream effectors such as RAS, RAF, andAKT that are associated with differential clinical outcomes.

The EGFR gene is present on chromosome 7p11.2 and has 28 exons codingfor a transmembrane receptor protein of 464 amino acids. Within EGFR,exons 5-7 and 13-16 code for the ligand binding domain while exons 18-24code for the TK domain. Autophosphorylation occurs in the region encodedby exons 25-28.

Mutations, amplifications or misregulations of EGFR or family membersare implicated in about 30% of all epithelial cancers. Althoughmutations can occur anywhere within the TK domain, a significant set ofEGFR mutations in lung cancer are observed in exons 18-21. The mostfrequent of these are in-frame deletions in exon 19 that occur inapproximately 45% of cases, followed by point mutations in exon 21, in40-45% of cases. While more than 20 different deletions are observed inexon 19, L858R in exon 21 is the most common point mutation detected. Denovo mutations are known to occur within EGFR that constitutively turnon the receptor. While the most important of these is T790M, a pointmutation in exon 20 that accounts for about 50% of cases, insertionalmutations in exon 20, which occur in about 5% of cases, have also beenassociated with resistance.

Mutations in KRAS at codons 12 and 13 occur in about 15-50% of NSCLCpatients, while BRAF mutations are detected in 1-2% of lung cancerpatients. KRAS and EGFR mutations appear to be mutually exclusive inNSCLC, with EGFR mutations occurring in non-smokers and KRAS mutationsin smokers. Approximately 30% to 50% of colorectal tumors are known tohave a mutated KRAS gene. A mutated BRAF gene, which is present in 5% to10% of tumors, may also affect response to anti-EGFR antibodies.

KRAS, NRAS, BRAF mutations. Multiple studies have shown that patientswith tumors harboring mutations in KRAS or NRAS exons 2, 3, or 4 predictlack of response to anti-EGFR antibody therapy given in combination withchemotherapy (Ciardiello et al. 2014; Douillard et al. 2013; Karthaus etal. 2013; Peeters et al. 2014; Stintzing et al. 2014; Tejpar et al.2014). Specific mutations of interest include KRAS mutations in codon 12or 13, for including without limitation KRAS c.34G>T (G12C); KRASc.34G>C (G12R); KRAS c.34G>A (G12S); KRAS c.35G>C (G12A); KRAS c.35G>A(G12D); KRAS c.35G>T (G12V); KRAS c.37G>T (G13C); KRAS c.37G>C (G13R);KRAS c.37G>A (G13S); KRAS c.38G>C (G13A); KRAS c.38G>A (G13D); NRASc.34G>T (G12C); NRAS c.34G>A (G12S); NRAS c.35G>C (G12A); NRAS c.35G>A(G12D); NRAS c.35G>T (G12V). The four most frequent KRAS mutations,G12D, G12V, G13D, G12C, account for 83% of all KRAS mutations. See, forexample, Peeters et al. JCO Feb. 20, 2013 vol. 31 no. 6 759-765; Stoltzeet al. Scientific Reports 5, Article number: 8535 (2015).

Various methods known in the art can be used for analysis of thegenotype of these genes. Traditional methods for detecting mutationsinvolved screening by direct DNA sequencing of the tumor tissue. Sangersequencing technology is available in most molecular diagnosticlaboratories, and it has the singular advantage of detecting alterationsacross a gene, including novel variants. Recent methodologies havefocused on targeted screening of mutations to achieve more rapid,robust, and sensitive tests. Molecular diagnostic laboratories currentlyuse a variety of methods, including amplification refractory mutationsystem, pyrosequencing, smart amplification process, high-resolutionmelting analysis, and restriction fragment length polymorphism, to namea few. These methods all distinguish between mutant and wild-type DNAwithin the region of interest. In contrast to direct sequencing, thelimit of detection for targeted analysis is ˜1-5% mutant DNA in thebackground of normal DNA.

Most laboratories use formalin-fixed, paraffin-embedded (FFPE) tissue totest for mutations. A few also use frozen tumor tissue. However,screening FFPE samples poses significant challenges, includingsuccessfully extracting the DNA, interferences from the fixatives usedfor embedding tissue, and most importantly, obtaining shorter ampliconsfor effective analysis, particularly if a PCR-based methodology is used.Alternate sample types such as fine needle aspirates and pleuraleffusions are currently being evaluated as viable options to enablequicker, easier diagnosis of malignancy. Micro-dissection of the tumorprior to testing is also helpful as it effectively enriches the sample,thereby increasing sensitivity.

A commercially available test for this purpose is therascreen KRAS RGQ(Rotor-Gene Q) PCR (polymerase chain reaction) Kit. Tests for mutationsin codons 12 or 13 of the KRAS gene can be performed on formalin-fixed,paraffin-embedded tissue from the primary tumor or a metastasis. PCRamplification and DNA sequence analysis or allele-specific PCR for BRAFV600E mutation status on formalin-fixed, paraffin-embedded tissue fromthe primary tumor or a metastasis

Anti-EGFR antibody. A number of clinically useful antibodies thatspecifically bind to and inhibit human EGFR are available and find usein the methods of the invention. Anti-EGFR monoclonal antibodies, forexample cetuximab, panitumumab, nimotuzumab, zalutumumab, and matuzumabbind to the extracellular domain of the EGFR monomer and compete forreceptor binding by the endogenous ligands, triggering receptorinternalization and blocking ligand-induced receptor activation.Cetuximab, which binds to the L2 domain of EGFR, is a chimeric proteinantibody composed of variable and constant regions from mouse and humansources, respectively, while panitumumab and nimotuzumab are human andhumanized antibodies. To date, the Food and Drug Administration hasapproved EGFR-targeted mAbs for use in advanced colorectal cancer,gliomas, and head and neck tumors.

The antibodies may be used as currently prescribed, or the dosage may bevaried to optimize the combination therapy. For example, cetuximab iscurrently prescribed at a dose of 400 mg/m² as an initial dose followedby 250 mg/m² weekly infusion. The dose may be reduced by 50% fornon-serious infusion reactions. Panitumumab is currently prescribed at 6mg/kg every 14 days as an intravenous infusion. The dose may be reducedby 50% for non-serious infusion reactions.

For example, anti-EGFR antibodies may be administered at a dose of fromabout 0.05 mg/kg, 0.1 mg/kg; 0.5 mg/kg, 1 mg/kg, 2.5 mg/kg, 5 mg/kg, 7.5mg/kg, 10 mg/kg, 12.5 mg/kg, 15 mg/kg, or more as required. Dosing maybe daily, every other day, semi-weekly, weekly, every 145 days, etc. fora period of time sufficient to achieve the desired result, e.g. fromabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more weeks. In someembodiments the dose for a combination therapy is lower than the doserequired for effectiveness as a monotherapy.

In addition to antibodies that directly inhibit EGFR signaling, thecombination methods of the present invention find use withnon-antagonistic antibodies, e.g. as described in international patentapplication WO2012058592 (herein specifically incorporated byreference); the non-antagonistic anti-EGFR antibody 13A9; and the like.

As used herein, “antibody” includes reference to an immunoglobulinmolecule immunologically reactive with a particular antigen, andincludes both polyclonal and monoclonal antibodies. The term alsoincludes genetically engineered forms such as chimeric antibodies (e.g.,humanized murine antibodies) and heteroconjugate antibodies. The term“antibody” also includes antigen binding forms of antibodies, includingfragments with antigen-binding capability (e.g., Fab′, F(ab′)₂, Fab, Fvand rIgG. The term also refers to recombinant single chain Fv fragments(scFv). The term antibody also includes bivalent or bispecificmolecules, diabodies, triabodies, and tetrabodies.

Selection of antibodies may be based on a variety of criteria, includingselectivity, affinity, cytotoxicity, etc. The phrase “specifically (orselectively) binds” to an antibody or “specifically (or selectively)immunoreactive with,” when referring to a protein or peptide, refers toa binding reaction that is determinative of the presence of the protein,in a heterogeneous population of proteins and other biologics. Thus,under designated immunoassay conditions, the specified antibodies bindto a particular protein sequences at least two times the background andmore typically more than 10 to 100 times background. In general,antibodies of the present invention bind antigens on the surface oftarget cells in the presence of effector cells (such as natural killercells or macrophages). Fc receptors on effector cells recognize boundantibodies.

An antibody immunologically reactive with a particular antigen can begenerated by recombinant methods such as selection of libraries ofrecombinant antibodies in phage or similar vectors, or by immunizing ananimal with the antigen or with DNA encoding the antigen. Methods ofpreparing polyclonal antibodies are known to the skilled artisan. Theantibodies may, alternatively, be monoclonal antibodies. Monoclonalantibodies may be prepared using hybridoma methods. In a hybridomamethod, an appropriate host animal is typically immunized with animmunizing agent to elicit lymphocytes that produce or are capable ofproducing antibodies that will specifically bind to the immunizingagent. Alternatively, the lymphocytes may be immunized in vitro. Thelymphocytes are then fused with an immortalized cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell.

Human antibodies can be produced using various techniques known in theart, including phage display libraries. Similarly, human antibodies canbe made by introducing of human immunoglobulin loci into transgenicanimals, e.g., mice in which the endogenous immunoglobulin genes havebeen partially or completely inactivated. Upon challenge, human antibodyproduction is observed, which closely resembles that seen in humans inall respects, including gene rearrangement, assembly, and antibodyrepertoire.

Antibodies also exist as a number of well-characterized fragmentsproduced by digestion with various peptidases. Thus pepsin digests anantibody below the disulfide linkages in the hinge region to produceF(ab)′₂, a dimer of Fab which itself is a light chain joined toV_(H)-C_(H1) by a disulfide bond. The F(ab)′₂ may be reduced under mildconditions to break the disulfide linkage in the hinge region, therebyconverting the F(ab)′₂ dimer into an Fab′ monomer. The Fab′ monomer isessentially Fab with part of the hinge region. While various antibodyfragments are defined in terms of the digestion of an intact antibody,one of skill will appreciate that such fragments may be synthesized denovo either chemically or by using recombinant DNA methodology. Thus,the term antibody, as used herein, also includes antibody fragmentseither produced by the modification of whole antibodies, or thosesynthesized de novo using recombinant DNA methodologies (e.g., singlechain Fv) or those identified using phage display libraries.

A “humanized antibody” is an immunoglobulin molecule which containsminimal sequence derived from non-human immunoglobulin. Humanizedantibodies include human immunoglobulins (recipient antibody) in whichresidues from a complementary determining region (CDR) of the recipientare replaced by residues from a CDR of a non-human species (donorantibody) such as mouse, rat or rabbit having the desired specificity,affinity and capacity. In some instances, Fv framework residues of thehuman immunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, a humanized antibody will comprise substantiallyall of at least one, and typically two, variable domains, in which allor substantially all of the CDR regions correspond to those of anon-human immunoglobulin and all or substantially all of the framework(FR) regions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

Antibodies of interest may be tested for their ability to induce ADCC(antibody-dependent cellular cytotoxicity) or ADCP (antibody dependentcellular phagocytosis). Antibody-associated ADCC activity can bemonitored and quantified through detection of either the release oflabel or lactate dehydrogenase from the lysed cells, or detection ofreduced target cell viability (e.g. annexin assay). Assays for apoptosismay be performed by terminal deoxynucleotidyl transferase-mediateddigoxigenin-11-dUTP nick end labeling (TUNEL) assay (Lazebnik et al.,Nature: 371, 346 (1994). Cytotoxicity may also be detected directly bydetection kits known in the art, such as Cytotoxicity Detection Kit fromRoche Applied Science (Indianapolis, Ind.).

A “patient” for the purposes of the present invention includes bothhumans and other animals, particularly mammals, including pet andlaboratory animals, e.g. mice, rats, rabbits, etc. Thus the methods areapplicable to both human therapy and veterinary applications. In oneembodiment the patient is a mammal, preferably a primate. In otherembodiments the patient is human.

The terms “subject,” “individual,” and “patient” are usedinterchangeably herein to refer to a mammal being assessed for treatmentand/or being treated. In an embodiment, the mammal is a human. The terms“subject,” “individual,” and “patient” encompass, without limitation,individuals having cancer. Subjects may be human, but also include othermammals, particularly those mammals useful as laboratory models forhuman disease, e.g. mouse, rat, etc.

The terms “cancer,” “neoplasm,” and “tumor” are used interchangeablyherein to refer to cells which exhibit autonomous, unregulated growth,such that they exhibit an aberrant growth phenotype characterized by asignificant loss of control over cell proliferation. Cells of interestfor detection, analysis, or treatment in the present application includeprecancerous (e.g., benign), malignant, pre-metastatic, metastatic, andnon-metastatic cells. Cancers of virtually every tissue are known. Thephrase “cancer burden” refers to the quantum of cancer cells or cancervolume in a subject. Reducing cancer burden accordingly refers toreducing the number of cancer cells or the cancer volume in a subject.The term “cancer cell” as used herein refers to any cell that is acancer cell or is derived from a cancer cell e.g. clone of a cancercell. Many types of cancers are known to those of skill in the art,including solid tumors such as carcinomas, sarcomas, glioblastomas,melanomas, lymphomas, myelomas, etc., and circulating cancers such asleukemias. Examples of cancer include but are not limited to, ovariancancer, breast cancer, colon cancer, lung cancer, prostate cancer,hepatocellular cancer, gastric cancer, pancreatic cancer, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, cancer of theurinary tract, thyroid cancer, renal cancer, carcinoma, melanoma, headand neck cancer, and brain cancer.

The “pathology” of cancer includes all phenomena that compromise thewell-being of the patient. This includes, without limitation, abnormalor uncontrollable cell growth, metastasis, interference with the normalfunctioning of neighboring cells, release of cytokines or othersecretory products at abnormal levels, suppression or aggravation ofinflammatory or immunological response, neoplasia, premalignancy,malignancy, invasion of surrounding or distant tissues or organs, suchas lymph nodes, etc.

As used herein, the terms “cancer recurrence” and “tumor recurrence,”and grammatical variants thereof, refer to further growth of neoplasticor cancerous cells after diagnosis of cancer. Particularly, recurrencemay occur when further cancerous cell growth occurs in the canceroustissue. “Tumor spread,” similarly, occurs when the cells of a tumordisseminate into local or distant tissues and organs; therefore tumorspread encompasses tumor metastasis. “Tumor invasion” occurs when thetumor growth spread out locally to compromise the function of involvedtissues by compression, destruction, or prevention of normal organfunction.

As used herein, the term “metastasis” refers to the growth of acancerous tumor in an organ or body part, which is not directlyconnected to the organ of the original cancerous tumor. Metastasis willbe understood to include micrometastasis, which is the presence of anundetectable amount of cancerous cells in an organ or body part which isnot directly connected to the organ of the original cancerous tumor.Metastasis can also be defined as several steps of a process, such asthe departure of cancer cells from an original tumor site, and migrationand/or invasion of cancer cells to other parts of the body.

The term “sample” with respect to a patient encompasses blood and otherliquid samples of biological origin, solid tissue samples such as abiopsy specimen or tissue cultures or cells derived therefrom and theprogeny thereof. The definition also includes samples that have beenmanipulated in any way after their procurement, such as by treatmentwith reagents; washed; or enrichment for certain cell populations, suchas cancer cells. The definition also includes sample that have beenenriched for particular types of molecules, e.g., nucleic acids,polypeptides, etc. The term “biological sample” encompasses a clinicalsample, and also includes tissue obtained by surgical resection, tissueobtained by biopsy, cells in culture, cell supernatants, cell lysates,tissue samples, organs, bone marrow, blood, plasma, serum, and the like.A “biological sample” includes a sample obtained from a patient's cancercell, e.g., a sample comprising polynucleotides and/or polypeptides thatis obtained from a patient's cancer cell (e.g., a cell lysate or othercell extract comprising polynucleotides and/or polypeptides); and asample comprising cancer cells from a patient. A biological samplecomprising a cancer cell from a patient can also include non-cancerouscells.

The term “diagnosis” is used herein to refer to the identification of amolecular or pathological state, disease or condition, such as theidentification of a molecular subtype of breast cancer, prostate cancer,or other type of cancer.

The term “prognosis” is used herein to refer to the prediction of thelikelihood of cancer-attributable death or progression, includingrecurrence, metastatic spread, and drug resistance, of a neoplasticdisease, such as ovarian cancer. The term “prediction” is used herein torefer to the act of foretelling or estimating, based on observation,experience, or scientific reasoning. In one example, a physician maypredict the likelihood that a patient will survive, following surgicalremoval of a primary tumor and/or chemotherapy for a certain period oftime without cancer recurrence.

As used herein, the terms “treatment,” “treating,” and the like, referto administering an agent, or carrying out a procedure, for the purposesof obtaining an effect. The effect may be prophylactic in terms ofcompletely or partially preventing a disease or symptom thereof and/ormay be therapeutic in terms of effecting a partial or complete cure fora disease and/or symptoms of the disease. “Treatment,” as used herein,may include treatment of a tumor in a mammal, particularly in a human,and includes: (a) preventing the disease or a symptom of a disease fromoccurring in a subject which may be predisposed to the disease but hasnot yet been diagnosed as having it (e.g., including diseases that maybe associated with or caused by a primary disease; (b) inhibiting thedisease, i.e., arresting its development; and (c) relieving the disease,i.e., causing regression of the disease.

Treating may refer to any indicia of success in the treatment oramelioration or prevention of an cancer, including any objective orsubjective parameter such as abatement; remission; diminishing ofsymptoms or making the disease condition more tolerable to the patient;slowing in the rate of degeneration or decline; or making the finalpoint of degeneration less debilitating. The treatment or ameliorationof symptoms can be based on objective or subjective parameters;including the results of an examination by a physician. Accordingly, theterm “treating” includes the administration of the compounds or agentsof the present invention to prevent or delay, to alleviate, or to arrestor inhibit development of the symptoms or conditions associated withcancer or other diseases. The term “therapeutic effect” refers to thereduction, elimination, or prevention of the disease, symptoms of thedisease, or side effects of the disease in the subject.

“In combination with”, “combination therapy” and “combination products”refer, in certain embodiments, to the concurrent administration to apatient of a first therapeutic and the compounds as used herein. Whenadministered in combination, each component can be administered at thesame time or sequentially in any order at different points in time.Thus, each component can be administered separately but sufficientlyclosely in time so as to provide the desired therapeutic effect.

“Concomitant administration” of a cancer therapeutic drug, ESA ortumor-directed antibody with a pharmaceutical composition of the presentinvention means administration with the high affinity CD47 reagent atsuch time that both the drug, ESA or antibody and the composition of thepresent invention will have a therapeutic effect. Such concomitantadministration may involve concurrent (i.e. at the same time), prior, orsubsequent administration of the drug, ESA or antibody with respect tothe administration of a compound of the invention. A person of ordinaryskill in the art would have no difficulty determining the appropriatetiming, sequence and dosages of administration for particular drugs andcompositions of the present invention.

As used herein, endpoints for treatment will be given a meaning as knownin the art and as used by the Food and Drug Administration.

Overall survival is defined as the time from randomization until deathfrom any cause, and is measured in the intent-to-treat population.Survival is considered the most reliable cancer endpoint, and whenstudies can be conducted to adequately assess survival, it is usuallythe preferred endpoint. This endpoint is precise and easy to measure,documented by the date of death. Bias is not a factor in endpointmeasurement. Survival improvement should be analyzed as a risk-benefitanalysis to assess clinical benefit. Overall survival can be evaluatedin randomized controlled studies. Demonstration of a statisticallysignificant improvement in overall survival can be considered to beclinically significant if the toxicity profile is acceptable, and hasoften supported new drug approval. A benefit of the methods of theinvention can include increased overall survival of patients.

Endpoints that are based on tumor assessments include DFS, ORR, TTP,PFS, and time-to-treatment failure (TTF). The collection and analysis ofdata on these time-dependent endpoints are based on indirectassessments, calculations, and estimates (e.g., tumor measurements).Disease-Free Survival (DFS) is defined as the time from randomizationuntil recurrence of tumor or death from any cause. The most frequent useof this endpoint is in the adjuvant setting after definitive surgery orradiotherapy. DFS also can be an important endpoint when a largepercentage of patients achieve complete responses with chemotherapy.

Objective Response Rate. ORR is defined as the proportion of patientswith tumor size reduction of a predefined amount and for a minimum timeperiod. Response duration usually is measured from the time of initialresponse until documented tumor progression. Generally, the FDA hasdefined ORR as the sum of partial responses plus complete responses.When defined in this manner, ORR is a direct measure of drug antitumoractivity, which can be evaluated in a single-arm study.

Time to Progression and Progression-Free Survival. TTP and PFS haveserved as primary endpoints for drug approval. TTP is defined as thetime from randomization until objective tumor progression; TTP does notinclude deaths. PFS is defined as the time from randomization untilobjective tumor progression or death. The precise definition of tumorprogression is important and should be carefully detailed in theprotocol.

As used herein, the term “correlates,” or “correlates with,” and liketerms, refers to a statistical association between instances of twoevents, where events include numbers, data sets, and the like. Forexample, when the events involve numbers, a positive correlation (alsoreferred to herein as a “direct correlation”) means that as oneincreases, the other increases as well. A negative correlation (alsoreferred to herein as an “inverse correlation”) means that as oneincreases, the other decreases.

“Dosage unit” refers to physically discrete units suited as unitarydosages for the particular individual to be treated. Each unit cancontain a predetermined quantity of active compound(s) calculated toproduce the desired therapeutic effect(s) in association with therequired pharmaceutical carrier. The specification for the dosage unitforms can be dictated by (a) the unique characteristics of the activecompound(s) and the particular therapeutic effect(s) to be achieved, and(b) the limitations inherent in the art of compounding such activecompound(s).

“Pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic, and desirable, and includes excipients that are acceptablefor veterinary use as well as for human pharmaceutical use. Suchexcipients can be solid, liquid, semisolid, or, in the case of anaerosol composition, gaseous.

“Pharmaceutically acceptable salts and esters” means salts and estersthat are pharmaceutically acceptable and have the desiredpharmacological properties. Such salts include salts that can be formedwhere acidic protons present in the compounds are capable of reactingwith inorganic or organic bases. Suitable inorganic salts include thoseformed with the alkali metals, e.g. sodium and potassium, magnesium,calcium, and aluminum. Suitable organic salts include those formed withorganic bases such as the amine bases, e.g., ethanolamine,diethanolamine, triethanolamine, tromethamine, N methylglucamine, andthe like. Such salts also include acid addition salts formed withinorganic acids (e.g., hydrochloric and hydrobromic acids) and organicacids (e.g., acetic acid, citric acid, maleic acid, and the alkane- andarene-sulfonic acids such as methanesulfonic acid and benzenesulfonicacid). Pharmaceutically acceptable esters include esters formed fromcarboxy, sulfonyloxy, and phosphonoxy groups present in the compounds,e.g., C₁₋₆ alkyl esters. When there are two acidic groups present, apharmaceutically acceptable salt or ester can be a mono-acid-mono-saltor ester or a di-salt or ester; and similarly where there are more thantwo acidic groups present, some or all of such groups can be salified oresterified. Compounds named in this invention can be present inunsalified or unesterified form, or in salified and/or esterified form,and the naming of such compounds is intended to include both theoriginal (unsalified and unesterified) compound and its pharmaceuticallyacceptable salts and esters. Also, certain compounds named in thisinvention may be present in more than one stereoisomeric form, and thenaming of such compounds is intended to include all single stereoisomersand all mixtures (whether racemic or otherwise) of such stereoisomers.

The terms “pharmaceutically acceptable”, “physiologically tolerable” andgrammatical variations thereof, as they refer to compositions, carriers,diluents and reagents, are used interchangeably and represent that thematerials are capable of administration to or upon a human without theproduction of undesirable physiological effects to a degree that wouldprohibit administration of the composition.

A “therapeutically effective amount” means the amount that, whenadministered to a subject for treating a disease, is sufficient toeffect treatment for that disease.

Methods of Use

Methods are provided for treating or reducing primary or metastaticcancer, specifically including EGFR-expressing epithelial cancers, e.g.adenocarcinomas, colorectal carcinomas; squamous cell carcinomas; basalcell carcinomas; ovarian cancer, pancreatic cancer, breast cancer,NSCLC; EGFR expressing gliomas; etc., in a regimen comprising contactingthe targeted cells with a combination of agents that (i) an agent thatblockades CD47 activity; and (ii) an antibody that specifically binds toEGFR. Such methods include administering to a subject in need oftreatment a therapeutically effective amount or an effective dose of thecombined agents of the invention, including without limitationcombinations of the reagent with a chemotherapeutic drug, radiationtherapy, or an ESA.

Effective doses of the combined agents of the present invention for thetreatment of cancer, vary depending upon many different factors,including means of administration, target site, physiological state ofthe patient, whether the patient is human or an animal, othermedications administered, and whether treatment is prophylactic ortherapeutic. Usually, the patient is a human, but nonhuman mammals mayalso be treated, e.g. companion animals such as dogs, cats, horses,etc., laboratory mammals such as rabbits, mice, rats, etc., and thelike. Treatment dosages can be titrated to optimize safety and efficacy.

In some embodiments, the therapeutic dosage of each agent may range fromabout 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the hostbody weight. For example dosages can be 1 mg/kg body weight or 10 mg/kgbody weight or within the range of 1-10 mg/kg. An exemplary treatmentregime entails administration once every two weeks or once a month oronce every 3 to 6 months. Therapeutic entities of the present inventionare usually administered on multiple occasions. Intervals between singledosages can be weekly, monthly or yearly. Intervals can also beirregular as indicated by measuring blood levels of the therapeuticentity in the patient. Alternatively, therapeutic entities of thepresent invention can be administered as a sustained releaseformulation, in which case less frequent administration is required.Dosage and frequency vary depending on the half-life of the polypeptidein the patient.

In prophylactic applications, a relatively low dosage may beadministered at relatively infrequent intervals over a long period oftime. Some patients continue to receive treatment for the rest of theirlives. In other therapeutic applications, a relatively high dosage atrelatively short intervals is sometimes required until progression ofthe disease is reduced or terminated, and preferably until the patientshows partial or complete amelioration of symptoms of disease.Thereafter, the patent can be administered a prophylactic regime.

In still other embodiments, methods of the present invention includetreating, reducing or preventing tumor growth, tumor metastasis or tumorinvasion of cancers including carcinomas, gliomas, etc. For prophylacticapplications, pharmaceutical compositions or medicaments areadministered to a patient susceptible to, or otherwise at risk ofdisease in an amount sufficient to eliminate or reduce the risk, lessenthe severity, or delay the outset of the disease, including biochemical,histologic and/or behavioral symptoms of the disease, its complicationsand intermediate pathological phenotypes presenting during developmentof the disease.

Compositions for the treatment of cancer can be administered byparenteral, topical, intravenous, intratumoral, oral, subcutaneous,intraarterial, intracranial, intraperitoneal, intranasal orintramuscular means. A typical route of administration is intravenous orintratumoral, although other routes can be equally effective.

Typically, compositions are prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid vehicles prior to injection can also be prepared.The preparation also can be emulsified or encapsulated in liposomes ormicro particles such as polylactide, polyglycolide, or copolymer forenhanced adjuvant effect, as discussed above. Langer, Science 249: 1527,1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997. Theagents of this invention can be administered in the form of a depotinjection or implant preparation which can be formulated in such amanner as to permit a sustained or pulsatile release of the activeingredient. The pharmaceutical compositions are generally formulated assterile, substantially isotonic and in full compliance with all GoodManufacturing Practice (GMP) regulations of the U.S. Food and DrugAdministration.

Toxicity of the combined agents described herein can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., by determining the LD₅₀ (the dose lethal to 50% of thepopulation) or the LD₁₀₀ (the dose lethal to 100% of the population).The dose ratio between toxic and therapeutic effect is the therapeuticindex. The data obtained from these cell culture assays and animalstudies can be used in formulating a dosage range that is not toxic foruse in human. The dosage of the proteins described herein liespreferably within a range of circulating concentrations that include theeffective dose with little or no toxicity. The dosage can vary withinthis range depending upon the dosage form employed and the route ofadministration utilized. The exact formulation, route of administrationand dosage can be chosen by the individual physician in view of thepatient's condition.

The pharmaceutical compositions can be administered in a variety of unitdosage forms depending upon the method of administration. For example,unit dosage forms suitable for oral administration include, but are notlimited to, powder, tablets, pills, capsules and lozenges. It isrecognized that compositions of the invention when administered orally,should be protected from digestion. This is typically accomplishedeither by complexing the molecules with a composition to render themresistant to acidic and enzymatic hydrolysis, or by packaging themolecules in an appropriately resistant carrier, such as a liposome or aprotection barrier. Means of protecting agents from digestion are wellknown in the art.

The compositions for administration will commonly comprise an antibodyor other ablative agent dissolved in a pharmaceutically acceptablecarrier, preferably an aqueous carrier. A variety of aqueous carrierscan be used, e.g., buffered saline and the like. These solutions aresterile and generally free of undesirable matter. These compositions maybe sterilized by conventional, well known sterilization techniques. Thecompositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, toxicity adjusting agents and thelike, e.g., sodium acetate, sodium chloride, potassium chloride, calciumchloride, sodium lactate and the like. The concentration of active agentin these formulations can vary widely, and will be selected primarilybased on fluid volumes, viscosities, body weight and the like inaccordance with the particular mode of administration selected and thepatient's needs (e.g., Remington's Pharmaceutical Science (15th ed.,1980) and Goodman & Gillman, The Pharmacological Basis of Therapeutics(Hardman et al., eds., 1996)).

Also within the scope of the invention are kits comprising thecompositions (e.g., anti-EGFR antibodies; anti-CD47 agents, andformulations thereof) of the invention and instructions for use. The kitcan further contain a least one additional reagent, e.g. achemotherapeutic drug, ESA, etc. Kits typically include a labelindicating the intended use of the contents of the kit. The term labelincludes any writing, or recorded material supplied on or with the kit,or which otherwise accompanies the kit.

The compositions can be administered for therapeutic treatment.Compositions are administered to a patient in an amount sufficient tosubstantially ablate targeted cells, as described above. An amountadequate to accomplish this is defined as a “therapeutically effectivedose.”, which may provide for an improvement in overall survival rates.Single or multiple administrations of the compositions may beadministered depending on the dosage and frequency as required andtolerated by the patient. The particular dose required for a treatmentwill depend upon the medical condition and history of the mammal, aswell as other factors such as age, weight, gender, administration route,efficiency, etc.

EXPERIMENTAL Example 1 Treatment of Colorectal Cancer with Combined CD47Blockade and Anti-EGFR

All methods have been described previously, which references are hereinspecifically incorporated by reference:

-   Dalerba, P., S. J. Dylla, Et Al. (2007). “Phenotypic    Characterization Of Human Colorectal Cancer Stem Cells.” Proc Natl    Acad Sci USA 104(24): 10158-10163.-   Weiskopf, K., A. M. Ring, Et Al. (2013). “Engineered Sirp alpha    Variants As Immunotherapeutic Adjuvants To Anticancer Antibodies.”    Science 341(6141): 88-91.-   Wllingham, S. B., J. P. Volkmer, Et Al. (2012). “The Cd47-Signal    Regulatory Protein Alpha (Sirpa) Interaction Is A Therapeutic Target    For Human Solid Tumors.” Proc Natl Acad Sci USA 109(17): 6662-6667.-   Epigentic and genetic features of 24 colon cancer cell lines, Ahmed    et al., Oncogenesis, 2013

Cancer Cells. DLD1 cells (ATCC), HT29 cells (ATCC), SW620 cells (ATCC),SW48 cells (ATCC), LS174T cells (ATCC), HCT116 cells (ATCC), and CACO-2cells (ATCC) were cultured in RPMI (ThermoFisher S.) (DLD1), EMEM(ThermoFisher S.) (CACO-2, LS174T), McCoy's 5A (ThermoFisher S.) (HT29,HCT116), or Leibovitz's L-15 (ThermoFisher S.) (SW48, SW 620)supplemented with 10% fetal bovine serum (Omega Scientific), 100 U/mLpenicillin and 100 μg/mL streptomycin (ThermoFisher S.). GFP-luciferase+DLD1 cell line was generated by transduction using a pCDH-CMV-MCS-EF1puro HIV-based lentiviral vector (Systems Biosciences) engineered toexpress an eGFP-luciferase2 (pgl4) fusion protein. Stable lines werecreated by sorting for GFP expression on FACSAria II cell sorters (BDBiosciences). UM8 patient xenograft cancer cells (adenocarcinoma,sigmoid colon, T3N0Mx) have been obtained from obtained from P. Dalerba,who established and characterized these previously (Dalerba, P., S. J.Dylla, Et Al. (2007). “Phenotypic Characterization Of Human ColorectalCancer Stem Cells.” Proc Natl Acad Sci USA 104(24): 10158-10163). Tumorcells were transduced overnight with lentivirus in culture mediacontaining 6 μg/mL polybrene. The following day, cells were washedrepeatedly to remove polybrene and extracellular lentivirus. Transduced(GFP+) cells were later isolated from xenograft tumors by FACS.

In Vitro Phagocytosis Assay. Peripheral blood mononuclear cells wereenriched by density gradient centrifugation and monocytes were purifiedwith anti-CD14 microbeads (Miltenyi) and differentiated to macrophagesby culture for 7-10 days in IMDM+GlutaMax (Invitrogen) supplemented with10% AB-Human Serum (Invitrogen) and 100 U/mL penicillin and 100 μg/mLstreptomycin (Invitrogen). Phagocytosis assays were performed byco-culture of 50,000 macrophages with 100,000 GFP+ tumor cells for 2hours, then analyzed using an LSRFortessa cell analyzer with highthroughput sampler (BD Biosciences). Antibodies used for treatmentincluded: IgG4 isotype control, anti-CD47-clone Hu5F9-G4 (Stanford), andanti-EGFR cetuximab (Bristoll-Myers Squibb). Macrophages were identifiedby flow cytometry using anti-CD206 antibody. Dead cells were excludedfrom the analysis by staining with DAPI (Sigma). Phagocytosis wasevaluated as the percentage of GFP+ macrophages and was normalized tothe maximal response by each independent donor against each cell line.

Mice. 6-8 week old NSG (NOD.Cg-Prkdcscid II2rgtmlWjIISzJ) mice wereused. The Stanford colony of these mice was founded by mice purchasedfrom Jackson Labs (Stock 005557).

In Vivo Imaging. Bioluminescent imaging was performed on an IVISSpectrum (Caliper Life Science) and quantified using Living Image 4.0software. D-luciferin (firefly) potassium salt (Biosynth) solution wasprepared by dissolving 1 g in 60 mL PBS. Mice were injected IP with 200μL luciferin solution. Total flux (photons/second) values were obtainedfrom the anatomic region of tumor engraftment twenty minutes afterluciferin injection.

Ex Vivo Detection of Metastases. Metastases to lungs and lymph nodeswere visualized by detecting GFP+ cancer cells in lungs and lymph nodeswith a M205 FA fluorescent dissecting microscope (Leica) fitted with aDFC 500 camera (Leica).

Xenograft Tumor Models. 50,000 GFP-luciferase+ DLD1 cells or 240,000GFP-luciferase+UM8 cells were transplanted with 50% Matrigel (BD) ontothe back of NSG mice. In both models, treatment was initiated uponconfirmation of tumor engraftment by bioluminescence imaging andcontinued as indicated. For all treatments, antibodies whereadministered by intraperitoneal injection (100 μl) as follows: PBS andHu5F9-G4 (250 μg) every other day; cetuximab (120 μg) and panitumumab(120 μg) once weekly. Tumor growth was monitored by bioluminescenceimaging. For the DLD1 model mice where analyzed for the presence of lungand lymph node metastases after completion of the indicated treatmentperiod by detection of GFP+ cancer cells in lungs and lymph nodes with afluorescent dissecting microscope.

Example 2 A Phase 1b/2 Trial of Hu5F9-G4 in Combination with Cetuximabin Solid Tumor and Advanced Metastatic Colorectal Cancer Patients

CRC is the third leading cause of cancer-related deaths in the US inwhich 20% of patients have metastatic disease. Treatment options arelimited for metastatic CRC patients after failing frontline treatment.Thus, novel and effective therapies are needed. Hu5F9-G4 is afirst-in-class monoclonal antibody targeting CD47, an anti-phagocyticsignal expressed on cancer stem cells, with anti-tumor efficacy achievedthrough depleting CRC and cancer stem cells by macrophage and T cellinduced elimination. Pre-clinical studies have demonstrated dramaticsynergistic anti-tumor effect when Hu5F9-G4 is combined with theclinically approved anti-EGFR monoclonal antibody cetuximab. Thissynergistic efficacy extends to KRAS mutant patients: which comprise upto 40% of all CRC patients, have limited therapeutic options, andwhereby single agent cetuximab in not FDA approved due to lack ofantitumor activity. Thus, Hu5F9-G4 can rescue and potentiate cetuximabactivity in this population with high unmet medical need. Thesepre-clinical data form the basis to test clinical anti-tumor proof ofconcept in the proposed Phase 1b/2 clinical trial in relapsed/refractoryKRAS mutant metastatic CRC and KRAS wildtype metastatic patientsrefractory to anti-EGFR therapies.

Metastatic colorectal adenocarcinoma is a tumor with high relapse rateand poor long-term survival. The EGFR targeting antibodies such ascetuximab and panitumumab have significantly improved prognosis in KRASwildtype patients. However, patients with KRAS mutations, which compriseapproximately 40-50% of colorectal cancers, do not respond to anti-EGFRantibody therapies. Thus, additional therapies are needed to addressthis high unmet medical need.

Hu5F9-G4 is a monoclonal antibody that targets CD47, an anti-phagocyticcell surface protein. Pre-clinical studies have demonstrated thatblockade of CD47 signaling through this antibody eliminates human tumorcells including colorectal cancer, through facilitating phagocytosis bymacrophages. Additional pre-clinical studies demonstrate that anti-CD47antibodies can synergize with Fc receptor-activating anti-cancerantibodies including cetuximab and panitumumab. Combination therapy withHu59-G4 and anti-EGFR antibodies has led to significant responses inboth RAS wildtype and mutant in vivo models compared to either agentalone.

The combination of Hu5F9-G4 and cetuximab is tested for safe tolerancein solid tumor patients, including colorectal cancer. A Phase 1b/2 trialestablishes the safety and tolerability and optimal dosing strategy ofHu5F9-G4 in combination with cetuximab administered intravenously toadvanced solid tumor patients and advanced metastatic colorectal cancerpatients. In the Phase 1b portion of the trial, the combination isevaluated in an all corner solid tumor population with an emphasis ontreating patients with CRC, head & neck, breast, pancreatic, and ovariancancer.

Patients at a starting cetuximab dose of 300 mg/m² followed by weeklydoses of 200 mg/m² are 25% and 20% reductions, respectively, from thefull single agent doses of cetuximab. The initial dose of Hu5F9-G4consists of a priming dose of 1 mg/kg followed by weekly maintenancedoses of 10 mg/kg. In the next cohort, cetuximab is escalated to thefull single agent dose of 400 mg/m² followed by weekly 250 mg/m².Subsequent dose cohorts escalate the dose of Hu5F9-G4 to the full singleagent dose including a loading dose strategy in the combination. Themaximum doses evaluated do not exceed the single agent recommended doseand schedule for each individual antibody. In the Phase 2 part of thetrial, preliminary antitumor activity is investigated at the recommendedPhase 2 dose of the combination in selected populations of CRC patients.

Key Inclusion Criteria

-   1. Adults≥18 years old-   2. Histological Diagnosis-   a. Phase 1b only: Histologically or cytologically confirmed advanced    solid malignancy with an emphasis on CRC, Head & Neck, breast,    pancreatic and ovarian cancers who have been treated with at least    one regimen of prior systemic therapy, or who refuse systemic    therapy, and for which there is no curative therapy available;-   b. Phase 2 RAS Mutant CRC: Histologically confirmed    advanced/metastatic RAS mutant CRC who have progressed or are    ineligible for both irinotecan- and oxaliplatin-based chemotherapy    OR-   c. Phase 2 RAS Wild-Type CRC: Histologically confirmed    advanced/metastatic RAS wildtype CRC who have progressed or are    ineligible for both irinotecan- and oxaliplatin-based chemotherapy    and who are relapsed or refractory to at least 1 prior systemic    therapy that included an anti-EGFR antibody, such as cetuximab,    panitumumab or others-   3. ECOG Score 0-2-   4. For the Phase 2 part only: Disease that is measurable or    assessable for response according to RECIST 1.1-   5. Laboratory measurements, blood counts:-   Hemoglobin≥9.5 g/dL-   ANC≥1.0×10⁹/mL-   Platelets≥75×10⁹/mL-   6. Laboratory measurements, hepatic function:-   AST/ALT≤5×ULN-   Bilirubin≤1.5×ULN or 3.0×ULN and primarily unconjugated if patient    has a documented history of Gilbert's syndrome or a genetic    equivalent-   7. Laboratory measurements, renal function:-   Serum creatinine 1.5×ULN or if elevated, a calculated GFR≤40    mL/min/1.73 m²-   8. Negative urine or serum pregnancy test within 30 days before    administration of Hu5F9-G4 for women of childbearing potential-   9. Females of childbearing potential must be willing to use 2    effective methods of contraception during the study and continue for    6 months after the last dose of study drug-   10. Males must be willing to use 1 highly effective method of    contraception during the study and continue for 6 months after the    last dose of study drug, if the partner is a female of childbearing    potential-   11. Subject has provided informed consent-   12. Must be willing and able to comply with the clinic visits and    procedures outlined in the study protocol-   13. Phase 2 only: Willing to consent to 1 mandatory pre-treatment    and 1 on-treatment tumor biopsy, unless determined to not be    feasible by the Investigator (reasons include, but are not limited    to, lack of accessible tumor tissue to biopsy and patient safety    issues)

Exclusion Criteria

-   1. Patients with active brain metastases (patients with stable    treated CNS lesions who are off corticosteroid and radiation therapy    for at least 3 weeks are not considered active)-   2. Prior anticancer therapy including chemotherapy, hormonal    therapy, or investigational agents within 2 weeks or within at least    4 half-lives prior to Hu5F9-G4 dosing (up to a maximum of 4 weeks),    whichever is longer. Localized non-CNS radiotherapy, pre-existing    hormonal therapy with LHRH agonists, low dose steroids (oral    prednisone or equivalent 20 mg per day), and treatment with    bisphosphonates and RANKL inhibitors are not criteria for exclusion.-   3. Prior treatment with CD47 or SIRPα-targeting agents-   4. Known active or chronic hepatitis B or C infection or HIV-   5. RBC transfusion dependence, defined as requiring more than 2    units of RBC transfusions during the 4-week period prior to the    first dose of Hu5F9-G4. RBC transfusions are permitted during    screening and prior to enrollment to meet the hemoglobin inclusion    criteria.-   6. History of hemolytic anemia or Evans syndrome in the last 3    months-   7. Phase 2 only: Second malignancy, except treated basal cell or    localized squamous skin carcinomas, or other malignancy that for    which treatment was completed at least 3 years ago and for which    there is no evidence of recurrence-   8. Significant medical diseases or conditions, as assessed by the    Investigators and Sponsor, that would substantially increase the    risk/benefit ratio of participating in the study. This includes, but    is not limited to, acute myocardial infarction within the last 6    months, unstable angina, uncontrolled diabetes mellitus, significant    active infections, severely immunocompromised state, and congestive    heart failure NYHA Class II-IV-   9. History of psychiatric illness or substance abuse likely to    interfere with ability to comply with protocol requirements or give    informed consent-   10. Pregnancy or active breast feeding-   11. Positive Direct Antiglobulin Test (DAT)

The objective response rate (ORR) of Hu5F9-G4 in combination withcetuximab in patients with RAS mutant and RAS wild-type CRC isdetermined. Secondary objectives include evaluation of thepharmacokinetic (PK) profile of Hu5F9-G4 in combination with cetuximab.In Phase 2 secondary parameters of efficacy are evaluated, such asduration of response (DOR), progression free survival (PFS), time totumor progression, and overall survival for patients with RAS mutant andRAS wild-type CRC treated with Hu5F9-G4 in combination with cetuximab.

Pharmacodynamic (PD) and predictive markers including immune cell subsetfrequencies and tumor penetration of Hu5F9-G4 in combination withcetuximab are assessed. Efficacy in molecular subtypes of CRC areassessed.

Hu5F9-G4 is a humanized monoclonal antibody against CD47 and cetuximabis a chimeric monoclonal antibody against EGFR. Both drugs areadministered intravenously. In Cycle 1, a priming dose of Hu5F9 isadministered on Day 1 and then weekly cetuximab and Hu5F9 areadministered with each cycle being 28 days. The first loading dose ofcetuximab is administered on day 8, followed by the first maintenancedose of Hu5F9-G4 on day 9 in Cycle 1 but all subsequent doses of thecombination are administered on the same starting on day 15. On days ofsimultaneous administration, cetuximab is infused first prior toHu5F9-G4 administration. This schedule is illustrated in the tablebelow:

Dose Dose Schedule (Day per 28-day Cycle) Cohort Drug/Dose (IV) Cycle 1Cycle 2+ 1 Hu5F9-G4 1 mg/kg (prime) Day 1 — Hu5F9-G4 10 mg/kg(maintenance) Day 9, 15, 22 Day 1, 8, 15, 27 Cetuximab 300 mg/m² (load)Day 8 Cetuximab 200 mg/m² (maintenance) Day 15, 22 Day 1, 8, 15, 22 2Hu5F9-G4 1 mg/kg (prime) Day 1 — Hu5F9-G4 10 mg/kg (maintenance) Day 9,15, 22 Day 1, 8, 15, 22 Cetuximab 400 mg/m² (load) Day 8 Cetuximab 250mg/m² (maintenance) Day 15, 22 Day 1, 8, 15, 22 3 Hu5F9-G4 1 mg/kg(prime) Day 1 — Hu5F9-G4 20 mg/kg (maintenance) Day 9, 15, 22 Day 1, 8,15, 22 Cetuximab 400 mg/m² (load) Day 8 Cetuximab 250 mg/m²(maintenance) Day 15, 22 Day 1, 8, 15, 22 4 ^(a) Hu5F9-G4 1 mg/kg(prime) Day 1 — Hu5F9-G4 20 mg/kg (load) Day 9 and 11 — Hu5F9-G4 10-20mg/kg Day 15, 22 Day 1, 8, 15, 22 (maintenance) Cetuximab 400 mg/m²(load) Day 8 — Cetuximab 250 mg/m² (maintenance) Day 15, 22 Day 1, 8,15, 22 ^(a) Potential loading dose cohort may be added if deemednecessary by the CTSC.

For Part A of the study, patients are treated for 8 weeks with Hu5F9-G4and cetuximab. DLT safety evaluation used for determination of MTD willoccur within the first 4 weeks. A response assessment (according tomodified RECIST criteria) will occur every 2 cycles. Response assessmentwill occur every two cycles from start of treatment until diseaseprogression.

Phase 2 includes two arms, one comprising patients with RAS mutant CRCand the other comprising patients with RAS wild type CRC. For the firststage of the Simon two-stage design, patients are treated for 8 weeksand then response rate assessed. Objective response rate (ORR) (CR+PR)is determined according to modified RECIST 1.1 criteria. Responseassessment will occur every two cycles from start of treatment untildisease progression. A maximum of 88 patients (44 patients per arm) areenrolled in Phase 2. Sample size for Phase 2 was based on a Simon's twostage minimax design for each cohort using a one sided alpha level of0.10 and a power of 0.80 based on a null hypothesis of 5% response ratecompared to an alternative hypothesis of 15% for each cohort.

Phase 1b total: 15 to 24 patients. Phase 2: Up to 88 patients total (44in each cohort) In each cohort: Simon Two-Stage design, Stage 1=29 pts;Stage 2=15 additional patients

Each publication cited in this specification is hereby incorporated byreference in its entirety for all purposes.

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, animal species or genera,and reagents described, as such may vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention, which will be limited only by the appended claims

As used herein the singular forms “a”, “and”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the culture” includes reference to one or more culturesand equivalents thereof known to those skilled in the art, and so forth.All technical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisinvention belongs unless clearly indicated otherwise.

What is claimed is:
 1. A method of targeting EGFR-expressing colorectalcarcinoma cells comprising an activating mutation in KRAS forimmunodepletion, the method comprising: testing the EGFR-expressingcolorectal carcinoma cells for the presence of an activating mutation inone or more of KRAS, NRAS and BRAF; selecting for treatment thecolorectal carcinoma cells in which one or more of said activatingmutations is present; contacting a population of the colorectalcarcinoma cells in an individual mammal in vivo with (i) an agent thatblockades CD47 activity selected from an antibody that binds to CD47; anantibody that binds to SIRPα; a soluble SIRPα-binding CD47 fragment; anda soluble CD47-binding SIRPα fragment; and (ii) an antibody thatspecifically binds to epidermal growth factor receptor; in a doseeffective to increase immunodepletion of the EGFR-expressing colorectalcarcinoma cells by phagocytic cells.
 2. The method of claim 1, whereinthe treatment provides for increased overall survival of the individual.3. The method of claim 1, wherein depletion of the target cells isenhanced relative to the depletion observed with a monotherapy of (i) anagent that blockades CD47 activity; or (ii) an anti-EGFR antibody. 4.The method of claim 1, wherein the agent that blockades CD47 activity isan anti-CD47 antibody.
 5. The method of claim 4, wherein the anti-CD47antibody comprises an IgG4 Fc region.
 6. The method according to claim1, wherein said mammal is a mouse.
 7. The method according to claim 1,wherein said mammal is a human.
 8. The method of claim 1, wherein theantibody that specifically binds to epidermal growth factor receptor isselected from the group consisting of cetuximab, panitumumab,nimotuzumab, zalutumumab and matuzumab.
 9. The method of claim 1,wherein the antibody that specifically binds to epidermal growth factorreceptor is other than an antibody conjugated to a drug.